Abstract

Background and Purpose Nitroglycerin, as well as nitric oxide, causes hyperpolarization and cGMP elevation in vascular smooth muscle cells. It is unknown whether nitroglycerin ameliorates vasospasm by an increase in cGMP levels after subarachnoid hemorrhage (SAH). The purpose of the present study was to measure the levels of both cGMP and cAMP in the cerebral arteries and parietal cerebral cortices in a primate model and to determine the effect of nitroglycerin on vasospasm after SAH.

Methods Chronic vasospasm was induced by clot placement around the right middle cerebral artery (MCA). Seven days after the surgery, angiography was repeated and either nitroglycerin (3 μg/kg per hour) or saline was administered intravenously. Angiography and regional cerebral blood flow (rCBF) measurements in the bilateral parietal cortices were performed before and after each treatment. Both cGMP and cAMP levels were measured in the cerebral arteries and bilateral parietal cortices.

Results A significant vasospasm occurred in the cerebral arteries on both sides, more prominently on the right side. Concomitantly, rCBF on the right side was significantly decreased (P<.05). In the right MCA, cGMP levels were significantly lower than in the normal MCA (P<.05). After the administration of nitroglycerin for 3 hours, the cerebral vessels were significantly dilated on both sides (P<.05), and rCBF was significantly increased on the right side (P<.05) but not on the left side. Although depressed cGMP levels in the right MCA were not recovered by nitroglycerin, a significant increase in cGMP levels was observed in the basilar artery (P<.05). In both parietal cortices, cGMP levels were significantly decreased after SAH (P<.05) and unchanged after nitroglycerin treatment. There were no significant changes in cAMP levels in SAH and after nitroglycerin treatment.

Conclusions The vasodilator effect of nitroglycerin in spastic MCA may not be mediated by an increase in cGMP levels, suggesting an involvement of hyperpolarization of the smooth muscle cells. Given the increase in rCBF, nitroglycerin may be therapeutic for the treatment of vasospasm.

Cyclic nucleotides have been thought of as second messengers in various tissues, including platelets and vascular smooth muscle cells.123 Particular attention has been paid to the intracellular levels of cAMP and cGMP 12 because both cAMP and cGMP are among the important intracellular messengers that can cause relaxation in vascular smooth muscle cells by different pathways.12 β-Adrenergic stimulators and prostacyclin, for example, relax vascular smooth muscle cells by elevating cAMP.13 Nitrovasodilators, the EDRF, and atriopeptins also relax the vasculature through cGMP-dependent mechanisms.2

Several investigators reported that in cerebral vasospasm after SAH, cGMP levels were decreased4 and cAMP levels actually increased.5 Although the exact mechanism was not given, the inhibition of spontaneously released EDRF and/or functional changes of smooth muscle cell membrane may account for this phenomenon. In a previous report, it was demonstrated that EDRF and the contractility of the primate MCA were significantly impaired in vasospasm and that an endothelium-independent relaxation mechanism remained to act as a potentially antagonist system to established smooth muscle contraction.6 In addition, an intracarotid infusion of NO increased CBF, decreased cerebral vascular resistance, reversed angiographic vasospasm, and decreased CBF velocity in the vasospastic artery.7 Therefore, we hypothesized that the cGMP production system, namely, the guanylate cyclase activation system, may continue working against vasospasm. Indeed, guanylate cyclase can be activated by a high concentration of hemoglobin, lipid peroxides, and hydroxyl radical, which may play an important role in the pathogenesis of vasospasm.28 We are unaware of any previous study of cAMP and cGMP levels in cerebral arteries and cortices after intravenous infusion of nitroglycerin in a primate model of SAH. In the present study, we investigated the effects of nitroglycerin on cerebral artery diameters and rCBF in such a model in correlation with the levels of cAMP and cGMP.

Materials and Methods

All protocols were evaluated and approved by the Animal Ethics Review Committee of the Mie University School of Medicine. The animals were cared for in accordance with the Guidelines for Animal Experiments in the Mie University School of Medicine.

Study Protocol

Twelve Japanese monkeys (Macaca fuscata) and four cynomolgus monkeys (M fascicularis), each weighing between 3.7 and 12.2 kg (mean±SD, 7.9±2.4 kg), were used. The monkeys were randomly assigned into three groups: nitroglycerin-treated (n=9), saline-treated (n=4), and control (n=3). On day 0, each monkey in the nitroglycerin- and saline-treated groups underwent cerebral angiography to determine the baseline vessel caliber before SAH induction. On day 7, cerebral angiography and rCBF measurements were undertaken to evaluate the degree of vasospasm. Either nitroglycerin (3 μg/kg per hour) or saline in the same volume was infused intravenously for 3 hours under monitoring of MABP, HR, ETCO2, and body temperature. During the administration of nitroglycerin or saline, cerebral angiography at 1 and 3 hours after the infusion and the rCBF measurements at 1, 2, and 3 hours after the infusion were repeated to evaluate the effect of nitroglycerin on vasospasm. SAH was not induced in the control group. After obtaining baseline cerebral angiography and the rCBF measurements, saline was infused for 3 hours intravenously at the same volume and rates as nitroglycerin. Angiography and the rCBF measurements were repeated by the same protocol as that of the nitroglycerin group. During the study, MABP, HR, and ETCO2 were recorded every 10 minutes. After the experiments, the animals were killed by exsanguination.

On day 0, the animals were anesthetized with an intramuscular injection of ketamine hydrochloride (6 to 10 mg/kg) and atropine sulfate (0.02 mg/kg). The animals were intubated, and a peripheral venous line was placed. An intravenous injection of sodium pentobarbital (20 mg/kg per hour) maintained anesthesia, and pancuronium bromide (0.05 mg/kg per hour) maintained paralysis. Tobramycin (2 mg/kg) was given intramuscularly. A 2:1 mixture of N2O/O2 on a ventilator (SN-480-3, Shimano Co) was used to maintain the ETCO2 level at approximately 40 mm Hg during continuous ETCO2 monitoring (POET 601, Criticare System, Inc). Body temperature was monitored by a rectal thermometer (TF-DN) and maintained at 37°C with a heating pad. With a sterile technique, the right axillary artery was dissected and catheterized with a 4F polyethylene catheter so that the tip was at the right innominate artery. The catheter was connected with a three-way stopcock to a pressure transducer (MK12030 US, Baxter). An arterial phase anteroposterior cerebral angiogram was obtained by manually injecting a contrast medium (10 mL of Iopamiron 300, Schering). On day 7, angiography was repeated before and at 1 and 3 hours after the start of each treatment.

Induction of SAH

After baseline cerebral angiography on day 0, ETCO2 was adjusted to approximately 40 mm Hg by controlled ventilation. The animal's head was placed in a three-point pin-fixation system, and a right frontotemporal craniectomy was performed with a sterile technique. Under an operating microscope, the dura mater was opened, and the temporal lobe was retracted posteriorly. The sylvian fissure was entered by sharp dissection, and the arachnoid membrane over the sphenoid segment of the MCA, the precommunicating segment of the ACA, and the ICA was opened. Between 2.5 to 7.0 mL of autologous arterial blood clot (4.3±1.4 mL) was placed around the exposed cerebral arteries. The dura was closed in a watertight fashion, and the temporal muscle and skin were sutured in layers. After the operation, paralysis was reversed by an intravenous administration of prostigmine methylsulfate (0.07 mg/kg) and atropine sulfate (0.02 mg/kg). The animals were extubated after they recovered their gag reflex. The animals were observed for development of operative complications and/or delayed neurological deficits.

Measurements of rCBF

rCBF was measured by a hydrogen clearance technique. On day 7, burr holes were made over both parietal bones. The head of the animal was fixed in a head frame, and platinum needle electrodes, 0.3 mm diameter (UHE-100, Unique Medical Co), were placed 20 mm lateral to the midline, 10 mm posterior to the coronal suture, and in the gray matter 2 mm below the brain surface. A reference Ag-AgCl electrode (UHE-001, Unique Medical) was placed under the scalp. Hydrogen gas was administered through the endotracheal tube for 2 minutes at a concentration of 10%. The clearance curve for the hydrogen concentration in the brain tissue was recorded (UR 2P, Unique Medical), and rCBF value was calculated by the initial-slope method (PHG-201, DDU-100, Unique Medical).91011

Drug Administration

After angiography and the rCBF measurements on day 7 were completed, each animal was treated with intravenous nitroglycerin at 3 μg/kg per hour or an equivalent volume of saline for 3 hours. Nitroglycerin (Millisrol, 0.5 mg/mL) was provided by Nippon Kayaku Co, Ltd (Tokyo, Japan).

Euthanasia of Animals and Preparation of Samples

The animals were killed with sodium pentobarbital (50 mg/kg IV) followed by transcranial perfusion with 0.5 L of saline under 100 mm Hg pressure to wash out the circulating blood. The brains were rapidly removed and immersed in Krebs-Henseleit solution equilibrated with 95% O2 and 5% CO2 at 4°C. The millimolar composition of the Krebs-Henseleit solution was as follows: NaCl 115.0, KCl 4.7, CaCl2 2.5, MgCl2 1.2, NaHCO3 25.0, KH2PO4 1.2, and glucose 10.0. A small quantity of bilateral parietal cortices was excised and immediately frozen in liquid nitrogen. The bilateral sphenoid segment of MCA and the basilar artery were separated from the brain, and the clots were carefully removed. The arteries were immediately frozen in liquid nitrogen. The samples were stored in the liquid nitrogen until measurements of cyclic nucleotides were made.

Measurements of Cyclic Nucleotides

The treatment of samples was performed according to the methods described previously with some modifications.12 The frozen arteries and cortices were pulverized individually with the use of an amalgamator (Amalgam mixer, GC Co) with a polytetrafluoroethylene capsule that contained a stainless steel ball, both of which were precooled in liquid nitrogen. The frozen fine powder that resulted was homogenized in ice-cold 6% trichloroacetic acid. The homogenate was then centrifuged at 5000g for 15 minutes at 4°C, and the supernatant was extracted with 3 volumes of water-saturated ether. Both cAMP and cGMP levels in the supernatant were measured by radioimmunoassay with a commercial kit (Yamasa Shoyu Co). The residue remaining after centrifugation of the homogenate was treated with 4 mol/L NaOH and used for the protein assay with a kit from Bio-Rad Lab. Both cAMP and cGMP levels were expressed as picomoles per milligram protein.

Radiological Assessment

During the angiography, exposure factors were maintained constant, and a radiopaque control standard was used for correction to constant magnification. Subtraction films of the anteroposterior projection were made. An experienced person who was unaware of the treatment groups measured the diameter of the intracranial cerebral arteries nine times with a calibrated optical micrometer (Scale Lupe No. 1983, PEAK) and determined a mean value. The arteries were measured bilaterally at the following points: sphenoid segment of the MCA, precommunicating segment of the ACA, intradural ICA (C1), cavernous portion of the ICA (C2), and the basilar artery.

Statistical Analysis

Data for cyclic nucleotides are expressed as mean±SE. Others are expressed as mean±SD. Comparisons within groups and at different times were made by paired t test, and intergroup comparisons at day 0 and day 7 were determined by one-way ANOVA. The level of significance of all tests of comparisons was P<.05.

Results

Neurological Deficits and Physiological Parameters

No monkeys developed delayed neurological deficits. When the mean values for body weight, volume of clots, MABP, HR, ETCO2, and body temperature measured at baseline and on day 7 were compared, there was no significant difference between the nitroglycerin- and saline-treated groups (Table 1⇓). In the control group, HR was significantly lower than in the SAH group (P<.05).

HR Changes

In the nitroglycerin group, HR was 130±8.0 bpm before administration (Table 2⇓). After the administration of nitroglycerin, HR was significantly increased at 1 hour (142±8.9 bpm; P<.05), 2 hours (144±12.3 bpm; P<.05), and 3 hours (147±12.0 bpm; P<.01). In the control group, HR at 3 hours after the administration of saline was significantly lower than the baseline value (P<.01).

MABP Changes

There was no significant difference in MABP among the three groups before the administration of nitroglycerin (Table 3⇓). After the administration of nitroglycerin, MABP was decreased significantly at 1 hour (91±11.0 mm Hg; P<.05), 2 hours (83±9.8 mm Hg; P<.001), and 3 hours (84±10.0 mm Hg; P<.01). In the saline and control groups, MABP did not change significantly over 3 hours.

Angiographic Vasospasm

All animals in the SAH group showed a significant vasospasm in the cerebral arteries on day 7 (Table 4⇓). Significant reductions in vessel caliber compared with baseline values were seen in the right MCA (P<.001), ACA (P<.001), C1 (P<.001), and C2 (P<.001). Also, a slight but significant vasospasm occurred in the left MCA (P<.001), C1 (P<.01), and C2 (P<.01). There was no significant difference in the degree of vasospasm between the nitroglycerin- and saline-treated groups before the administration of nitroglycerin on day 7. After the administration of nitroglycerin for 1 hour, the vessel caliber of the left ACA and the right C2 were significantly increased, from 96% to 105% of baseline value (P<.05) and from 77% to 84% (P<.05), respectively (Table 4⇓). After the administration of nitroglycerin for 3 hours, the vessel caliber on both sides and in the basilar artery were significantly increased. Although the increases in vessel caliber were significant, the vessels did not reach the normal caliber on the right side. On the left side, the vessel calibers were increased up to the baseline value after being treated with nitroglycerin for 3 hours. In the saline and control groups, there were no significant changes in vessel caliber over the course of 3 hours of treatment.

rCBF Changes

In the nitroglycerin group, before the administration of nitroglycerin, rCBF on the right side was significantly lower than that on the left side (P<.05) (Fig 1⇓). After the administration of nitroglycerin for 1 hour, the right-side rCBF was significantly increased (P<.05). Over the course of a 3-hour administration of nitroglycerin, rCBF on the right side was maintained at the same level as that on the left side. rCBF on the left side was also increased during the 3 hours, but not significantly. In the saline group, rCBF on the right side was significantly lower than that on the left side before the administration (P<.05) (Fig 1⇓). rCBF on both sides was unchanged during the administration of saline. In the control group, rCBF was the same on both sides before and after the administration of saline (data not shown).

rCBF changes before and after treatment with nitroglycerin (top) or saline (bottom). pre indicates pretreatment; hr, hours after each treatment. *P<.05, significant difference from the contralateral side.

cGMP Levels in Cerebral Arteries and Cerebral Cortices

In the control group, basal cGMP levels in the right MCA, left MCA, and the basilar artery were at similar levels: 13.9±1.5 pmol/mg protein, 13.4±2.8 pmol/mg protein, and 12.2±1.3 pmol/mg protein, respectively (Fig 2⇓). In the saline group, cGMP in the right MCA was significantly decreased compared with the control group (P<.05). In the nitroglycerin group, cGMP in the left MCA and the basilar artery was approximately three times higher than in the saline group. The increase was significant in the basilar artery (P<.05). However, nitroglycerin did not increase cGMP in the right MCA. In the control group, basal cGMP levels in the right and left parietal cortices were 2.9±0.3 pmol/mg protein and 3.1±0.2 pmol/mg protein, respectively (Fig 2⇓). In the saline group, cGMP in each cortex was significantly decreased compared with the control group (P<.05). Cortical cGMP levels on the left side were increased in the nitroglycerin group, but not significantly.

cGMP levels in the cerebral arteries (top) and cerebral cortices (bottom). In the saline group, cGMP levels in the right (R.) MCA were significantly lower than in the control group (*P<.05). In the nitroglycerin group (GTN), cGMP levels in the basilar artery (BA) were significantly increased compared with the saline group (*P<.05). In the saline group, cGMP levels in both parietal cortices were significantly lower than in the control group (*P<.05). L. indicates left; Cx, parietal cortex.

cAMP Levels in Cerebral Arteries and Cerebral Cortices

There were no significant differences in cAMP levels among treatment groups (Table 5⇓).

Discussion

The major findings of this study were as follows: (1) significant vasospasm occurred in cerebral arteries on day 7, and vasospasm was attenuated after nitroglycerin administration; (2) rCBF on the right side was significantly decreased after SAH, and nitroglycerin significantly improved rCBF on the same side; (3) cGMP levels were significantly decreased in the right MCA and cerebral cortices after SAH, and nitroglycerin did not increase those cGMP levels; (4) nitroglycerin significantly increased cGMP levels in the basilar artery; and (5) cAMP levels were not affected by either SAH or nitroglycerin.

In this study, MABP was significantly decreased by nitroglycerin, but rCBF was increased. In the same primate model, the central conduction time on the clot side has been observed to remain fairly constant in MABPs above 80 mm Hg.13 The change in rCBF was found to be very small in MABPs of 80 to 100 mm Hg.13 Thus, the dosage of nitroglycerin in our study would be safe for the maintenance of rCBF and neuronal function against systemic hypotension. In patients with vasospasm, caution should be exercised against an excessive decrease in blood pressure by nitroglycerin. In such cases, dopamine and phenylephrine are preferable for hypertension-hypervolemic treatment.1415

In dogs, nitroglycerin has a potent vasodilative effect by releasing NO, particularly in large-capacitance arteries, with relatively little effect on small intraparenchymal resistance arteries that control CBF.14 Although some experimental evidence suggests that a large, rapid, “bolus” intravenous infusion of nitroglycerin (200 μg) transiently raises intracranial pressure, a prolonged continuous intravenous infusion of nitroglycerin (5 μg/kg per minute) does not alter normal intracranial pressure or CBF.14 In the normal monkey, intravenous nitroglycerin at rates of 5 μg/kg per minute dilates the large-capacitance vessels of the brain without adversely affecting rCBF or intracranial pressure.15 When the MABP was increased to 12% of control values by phenylephrine, rCBF did not change.15 In our study, despite a fall in MABP, rCBF remained stable during the 3 hours of nitroglycerin infusion. On the right side, rCBF was significantly increased after 1 hour of nitroglycerin infusion. It can be inferred that the infusion rate of 3 μg/kg per minute of nitroglycerin effectively improved the rCBF without an adverse effect on intracranial pressure and may thus be safe for patients with SAH.

It is well known that cGMP levels are very low in vasospasm,4 since the expression of soluble guanylate cyclase was reduced in a canine two-hemorrhage model of SAH.16 In addition, high-energy phosphates such as guanosine triphosphate, the substrate for production of cGMP, were significantly reduced in the spastic basilar arteries.17 In the present study, nitroglycerin dilated spastic cerebral arteries and improved rCBF, which had been depressed by SAH. The possible explanations for this are: (1) improvement of vasospasm in large pial arteries by nitroglycerin may lead to an increase in rCBF; (2) nitroglycerin may recruit collateral blood vessels; (3) nitroglycerin may activate sensory fibers to release calcitonin gene–related peptide, which relaxes cerebral vascular smooth muscle,18 by activating ATP-sensitive K+ channels19 ; and (4) NO and nitrovasodilators can hyperpolarize smooth muscle of several different isolated blood vessels, including the cerebral artery.20 Our results suggest that the vasodilation effects of nitroglycerin in vasospasm may be mediated by the activation of K+ channels rather than by an increase in cGMP levels.

It is known that in the central nervous system, the excitatory neurotransmitter glutamate can elicit large increases in cGMP levels.21 It was recently demonstrated that extracellular glutamate and aspartate concentrations rose to very high levels in the medial temporal and subfrontal cortex after surgery for SAH and aneurysm.22 These increased levels of excitatory amino acids correlated well with the clinical course and neurological symptoms of the patients.22 If there was an increase in glutamate levels in the parietal cortex, increased levels of cGMP would be expected in the primate model used in the present study. However, cGMP levels in both parietal cortices were significantly reduced. It was postulated that levels of excitatory amino acids might not increase in the parietal cortices, and/or that the inhibition of postsynaptic guanylate cyclase by oxyhemoglobin might be involved.16 Although nitroglycerin did not increase cGMP levels in the parietal cortices, nitroglycerin may have the additional effect of preventing N-methyl-d-aspartate receptor–mediated neurotoxicity by aiding NO group transfer to thiol of the redox modulatory site of the N-methyl-d-aspartate receptor.23

In contrast to the results of a previous study,5 we observed that cAMP levels were not affected by either SAH or nitroglycerin. Although the exact explanation for this event cannot be given here, differences in animal species and differences in the time courses by which cAMP was determined may account for this discrepancy.

In conclusion, nitroglycerin may have a beneficial effect on both arterial diameter and rCBF without adverse effects on vasospasm.

Selected Abbreviations and Acronyms

ACA

=

anterior cerebral artery

bpm

=

beats per minute

CBF

=

cerebral blood flow

EDRF

=

endothelium-derived relaxing factor

ETCO2

=

end-tidal CO2

HR

=

heart rate

ICA

=

internal carotid artery

MABP

=

mean arterial blood pressure

MCA

=

middle cerebral artery

NO

=

nitric oxide

rCBF

=

regional cerebral blood flow

SAH

=

subarachnoid hemorrhage

Acknowledgments

This study was supported by grants from the Nippon Kayaku Co (Dr Kanamaru). We thank Dr Isao Yamamoto for valuable technical advice on the measurement of cyclic nucleotides and Dr Loch R. Macdonald for critical review of the manuscript.

Editorial Comment

Existing evidence suggests that decreased availability of NO and impaired production of its second messenger, cGMP, may play very important roles in the development of chronic cerebral vasospasm after SAH.1R2R3R4R Consistent with this concept are findings demonstrating that experimental vasospasm could be reversed by intravenous administration of glycerol trinitrate,5R intracarotid infusion of NO,6R or restoration of endogenous NO production in the arterial wall by administration of l-arginine and superoxide dismutase.7R

cGMP is not an exclusive mediator of NO-induced vasodilatation. In peripheral arteries, vasodilatation to NO is in part mediated by calcium-activated potassium channels.8R This finding has important implications for understanding the vasodilator effect of NO in normal and diseased cerebral arteries. In the study performed on the primate model of cerebral vasospasm, Nakao et al demonstrated that intravenous administration of nitroglycerin caused vasodilatation of spastic arteries despite the fact that there was no significant increase in cGMP levels. This is the first in vivo study designed to simultaneously measure the vasodilator effect of nitroglycerin and cGMP production in monkeys with developed vasospasm. The most important implication of the presented findings is that nitrovasodilators and NO itself may indeed produce vasodilatation by mechanisms that do not require activation of guanylate cyclase. The cGMP-independent mechanisms responsible for mediation of NO-induced relaxations in normal and vasospastic arteries remain to be determined. Interestingly, increased vasodilator reactivity of spastic arteries to the ATP-sensitive potassium channel activator aprikalim has been detected in rats,9R supporting the idea that activation of potassium channels could become an important mechanism of vasodilatation in diseased arteries. Whether in human vascular tissue NO may activate potassium channels independently of cGMP production, and whether this vasodilator mechanism is upregulated in cerebral arteries with developed vasospasm, remain attractive questions for future investigation.